Andrew P. Mizisin

Evolution of Neuropathologic Abnormalities Associated with Blood-Brain Barrier Breakdown in Transgenic Mice Expressing Interleukin-6 in Astrocytes

Abstract. As both astrocytes and cytokines modulate the permeability of cerebral endothelial cells, transgenic animal models which overexpress cytokines, such as interleukin-6 (IL-6), may provide insight into the neuropathological consequences of increased BBB permeability. In this study, a GFAP-IL6 transgenic mouse model and horseradish peroxidase (HRP) were used to investigate BBB permeability and associated neuropathologic changes. In the cerebellum of control mice, the BBB developed between postnatal days 7 and 14. In transgenic mice, the BBB never developed and extensive breakdown was evident in both high- and low-expressor animals by 1 month after birth. Vascular proliferation was apparent from birth in association with development and retention of normal cerebellar architecture until 3 and 6 months in high- and low-expressor animals, respectively. At these times, a leptomeningeal inflammatory infiltrate, vacuolated astrocytic foot processes and endothelial abnormalities were apparent in the cerebellum. At 6 months in high-expressor and 12 months in low-expressor animals, parenchymal inflammation, gliosis, spongiform change, axonal degeneration and macrophage accumulation were evident. The findings suggest that increased production of IL-6 can influence the development and physiologic function of the BBB as well as contribute to parenchymal central nervous system injury.

Prevention of sensory disorders in diabetic Sprague-Dawley rats by aldose reductase inhibition or treatment with ciliary neurotrophic factor

Aims/hypothesisSensory neuropathy in diabetic patients frequently presents itself as progressive loss of thermal perception, while some patients describe concurrent spontaneous pain, allodynia or hyperalgesia. Diabetic rats develop thermal hypoalgesia and tactile allodynia by unknown mechanisms. We investigated whether sensory disorders in rats were related to glucose metabolism by aldose reductase. We also explored the therapeutic potential of exogenous neurotrophic factors.MethodsBehavioural assessments of thermal and tactile sensitivity were performed in normal rats and in rats with streptozotocin-induced diabetes. Some of the rats were treated with insulin, aldose reductase inhibitors, ciliary neurotrophic factor or brain-derived neurotrophic factor.ResultsThermal hypoalgesia was present after 8 weeks of diabetes and was prevented by insulin treatment, which maintained normoglycaemia, by the aldose reductase inhibitor Statil or by ciliary neurotrophic factor. Brain-derived neurotrophic factor did not have an effect. When diabetic rats were tested after shorter durations of diabetes, they showed transient thermal hyperalgesia after 4 weeks which progressed to thermal hypoalgesia after 8 weeks. The aldose reductase inhibitor IDD 676 (Lidorestat), given from the onset of diabetes, prevented the development of thermal hyperalgesia and also stopped progression to thermal hypoalgesia when delivered in the last 4 weeks of an 8-week period of diabetes. Tactile allodynia was not prevented by neurotrophic factor or aldose reductase inhibitor treatment.Conclusions/interpretationTransient thermal hyperalgesia and subsequent progressive thermal hypoalgesia occur in diabetic rats secondary to exaggerated flux through the polyol pathway. A depletion of ciliary neurotrophic factor mediated by the polyol pathway may be involved in the aetiology of thermal hypoalgesia.

Reactive, degenerative, and proliferative Schwann cell responses in experimental galactose and human diabetic neuropathy

Abstract Despite early descriptions of hypertrophic Schwann cells and onion-bulb formation in patients with diabetic neuropathy, clinical and experimental studies have emphasized axonal pathology. In recent years, the Schwann cell has been further implicated in diabetic neuropathy because it is the primary intrafascicular location for the first enzyme of the polyol pathway, aldose reductase, which appears to have a role in modulating a variety of complications of diabetes, including diabetic neuropathy. To further explore the role of polyol pathway flux in the pathogenesis of Schwann cell injury, ultrastructural abnormalities of Schwann cells in human diabetic neuropathy (HDN) were compared with those in experimental galactose neuropathy (EGN), a well-characterized model of hyperglycemia without hypoinsulinemia. Similar to previous studies of EGN, reactive, degenerative and proliferative changes of Schwann cells were observed after 2, 4 and 24 months of galactose intoxication. Reactive changes included accumulation of lipid droplets, π granules of Reich and glycogen granules, increased numbers of subplasmalemmal vesicles, cytoplasmic expansion, and capping. Degenerative changes included enlargement of mitochondria and effacement of cristae, and disintegration of both abaxonal and adaxonal cytosol and organelles. Both demyelination and onion-bulb formation were seen at all time points, although supernumerary Schwann cells and axonal degeneration were most numerous after 24 months of galactose feeding. In sural nerve biopsy samples from patients with diabetes and progressive worsening of neuropathy, ultrastructural abnormalities in Schwann cells encompassed the full range of reactive, degenerative and proliferative changes described in galactose-fed rats. The concordance of fine-structural observations in nerves from galactose-fed rats and these adult-onset diabetic patients emphasizes the role of flux through aldose reductase in the complex pathology of diabetic neuropathy and points to the utility of galactose intoxication in helping to understand this metabolic disorder.

Reduced heat sensitivity and epidermal nerve fiber immunostaining following single applications of a high-concentration capsaicin patch

Abstract Capsaicin‐containing plant extracts have been used as topical treatments for a variety of pain syndromes for many centuries. Current products containing capsaicin in low concentrations (usually 0.025–0.075% w/w) have shown efficacy against a variety of pain conditions in clinical studies. However, in order to produce significant analgesic effects, these formulations require frequent re‐dosing, often as much as three to five times daily for several weeks. Previous functional and immunohistochemical studies following prolonged exposures to low‐concentration capsaicin cream suggested that the duration and onset of analgesic efficacy correlate with a reduction of cutaneous nociceptive sensory nerve fiber responsiveness and immunostaining. The purpose of the present study was to determine whether a single topical application of a high‐concentration capsaicin‐containing (8% w/w) patch for 120 min or less would induce similar effects on cutaneous nociceptive nerve fibers. Seven days following patch application, changes in heat and cold perception thresholds were determined by quantitative sensory testing and punch biopsies were collected to assess epidermal nerve fiber (ENF) immunostaining density at the application site using PGP 9.5 as a marker. The results show a significant reduction of heat, but not cold, sensitivity and reduction of ENF immunostaining with high‐capsaicin concentration patch applications for 60 or 120 min, compared to placebo patch applications. Application sites exposed to low‐capsaicin concentration (0.04% w/w) patches for 120 min or high‐concentration patches for 30 min were not significantly different from placebo with respect to either thermal threshold detection or ENF immunostaining. The ability of a single 60 min high‐concentration patch application to mimic effects produced by prolonged exposure to low‐concentration capsaicin creams suggests a new approach to the management of chronic pain syndromes.

Nesprin 1 is critical for nuclear positioning and anchorage

Nesprin 1 is an outer nuclear membrane protein that is thought to link the nucleus to the actin cytoskeleton. Recent data suggest that mutations in Nesprin 1 may also be involved in the pathogenesis of Emery-Dreifuss muscular dystrophy. To investigate the function of Nesprin 1 in vivo, we generated a mouse model in which all isoforms of Nesprin 1 containing the C-terminal spectrin-repeat region with or without KASH domain were ablated. Nesprin 1 knockout mice are marked by decreased survival rates, growth retardation and increased variability in body weight. Additionally, nuclear positioning and anchorage are dysfunctional in skeletal muscle from knockout mice. Physiological testing demonstrated no significant reduction in stress production in Nesprin 1-deficient skeletal muscle in either neonatal or adult mice, but a significantly lower exercise capacity in knockout mice. Nuclear deformation testing revealed ineffective strain transmission to nuclei in muscle fibers lacking Nesprin 1. Overall, our data show that Nesprin 1 is essential for normal positioning and anchorage of nuclei in skeletal muscle.

MTM1 mutation associated with X-linked myotubular myopathy in Labrador Retrievers.

Mutations in the MTM1 gene encoding myotubularin cause X-linked myotubular myopathy (XLMTM), a well-defined subtype of human centronuclear myopathy. Seven male Labrador Retrievers, age 14–26 wk, were clinically evaluated for generalized weakness and muscle atrophy. Muscle biopsies showed variability in fiber size, centrally placed nuclei resembling fetal myotubes, and subsarcolemmal ringed and central dense areas highlighted with mitochondrial specific reactions. Ultrastructural studies confirmed the centrally located nuclei, abnormal perinuclear structure, and mitochondrial accumulations. Wild-type triads were infrequent, with most exhibiting an abnormal orientation of T tubules. MTM1 gene sequencing revealed a unique exon 7 variant in all seven affected males, causing a nonconservative missense change, p.N155K, which haplotype data suggest derives from a recent founder in the local population. Analysis of a worldwide panel of 237 unaffected Labrador Retrievers and 59 additional control dogs from 25 other breeds failed to identify this variant, supporting it as the pathogenic mutation. Myotubularin protein levels and localization were abnormal in muscles from affected dogs, and expression of GFP-MTM1 p.N155K in COS-1 cells showed that the mutant protein was sequestered in proteasomes, where it was presumably misfolded and prematurely degraded. These data demonstrate that XLMTM in Labrador Retrievers is a faithful genetic model of the human condition.

Epidermal Nerve Fiber Quantification in the Assessment of Diabetic Neuropathy

Assessment of cutaneous innervation in skin biopsies is emerging as a valuable means of both diagnosing and staging diabetic neuropathy. Immunolabeling, using antibodies to neuronal proteins such as protein gene product 9.5, allows for the visualization and quantification of intraepidermal nerve fibers. Multiple studies have shown reductions in intraepidermal nerve fiber density in skin biopsies from patients with both type 1 and type 2 diabetes. More recent studies have focused on correlating these changes with other measures of diabetic neuropathy. A loss of epidermal innervation similar to that observed in diabetic patients has been observed in rodent models of both type 1 and type 2 diabetes and several therapeutics have been reported to prevent reductions in intraepidermal nerve fiber density in these models. This review discusses the current literature describing diabetes-induced changes in cutaneous innervation in both human and animal models of diabetic neuropathy.

Homeostatic regulation of the endoneurial microenvironment during development, aging and in response to trauma, disease and toxic insult

The endoneurial microenvironment, delimited by the endothelium of endoneurial vessels and a multi-layered ensheathing perineurium, is a specialized milieu intérieur within which axons, associated Schwann cells and other resident cells of peripheral nerves function. The endothelium and perineurium restricts as well as regulates exchange of material between the endoneurial microenvironment and the surrounding extracellular space and thus is more appropriately described as a blood–nerve interface (BNI) rather than a blood–nerve barrier (BNB). Input to and output from the endoneurial microenvironment occurs via blood–nerve exchange and convective endoneurial fluid flow driven by a proximo-distal hydrostatic pressure gradient. The independent regulation of the endothelial and perineurial components of the BNI during development, aging and in response to trauma is consistent with homeostatic regulation of the endoneurial microenvironment. Pathophysiological alterations of the endoneurium in experimental allergic neuritis (EAN), and diabetic and lead neuropathy are considered to be perturbations of endoneurial homeostasis. The interactions of Schwann cells, axons, macrophages, and mast cells via cell–cell and cell–matrix signaling regulate the permeability of this interface. A greater knowledge of the dynamic nature of tight junctions and the factors that induce and/or modulate these key elements of the BNI will increase our understanding of peripheral nerve disorders as well as stimulate the development of therapeutic strategies to treat these disorders.

Fine-structural localization of aldose reductase and ouabain-sensitive, K+-dependent p-nitro-phenylphosphatase in rat peripheral nerve

SummaryAldose reductase was visualized by light and electron microscopy using a goat anti-rat antibody with immunoperoxidase and immunogold, respectively. Ouabain-sensitive, K+-dependent, p-nitro-phenylphosphatase, a component of (Na+, K+)-ATPase, was localized at the electron microscopic level by enzyme histochemistry using p-nitro-phenylphosphate as substrate. In peripheral nerve, spinal ganglia and roots, the Schwann cell of myelinated fibers was the principal site of aldose reductase localization. Immunostaining was intense in the paranodal region and the Schmidt-Lanterman clefts as well as in cytoplasm of the terminal expansions of paranodal myelin lamellae and the nodal microvilli. Schwann cell cytoplasm of unmyelinated fibers were faintly labelled. Endoneurial vessel endothelia, pericytes and perineurium failed to bind appreciable amounts of aldose reductase antibody. However, mast cell granules bound antibody strongly. In contrast, p-nitro-phenylphosphatase reaction product was detected in the nodal axolemma, terminal loops of Schwann cell cytoplasm and the innermost layer of perineurial cells. In endothelial cells, reaction product was localized on either the luminal or abluminal, or on both luminal and abluminal plasmalemma. Endothelial vesicular profiles were often loaded with reaction product. Occasional staining of myelin and axonal organelles was noted. Mast cells lacked reaction product.

Dissociation of thermal hypoalgesia and epidermal denervation in streptozotocin-diabetic mice☆

The quantification of epidermal innervation, which consists primarily of heat-sensitive C-fibers, is emerging as a tool for diagnosing and staging diabetic neuropathy. However, the relationship between changes in heat sensitivity and changes in epidermal innervation has not yet been adequately explored. Therefore, we assessed epidermal nerve fiber density and thermal withdrawal latency in the hind paw of Swiss Webster mice after 2 and 4 weeks of streptozotocin-induced diabetes. Thermal hypoalgesia developed after only 2 weeks of diabetes, but a measurable reduction in PGP9.5-immunoreactive epidermal nerve fiber density did not appear until 4 weeks. These data suggest that impaired epidermal nociceptor function contributes to early diabetes-induced thermal hypoalgesia prior to the loss of peripheral terminals.